Protein Analysis

• Ion exchange chromatography separates proteins based on their net charge, with positively charged proteins binding to a negatively charged resin and vice versa.
• Proteins are the most abundant macromolecules in the biological system
• Detecting proteins as biomarkers are useful in diagnosing diseases
• Column Chromatography is a way of separating protein molecules depending on their specific differences in physical-chemical characteristics
• Gel Permeation/Gel Filtration/Size Exclusion Chromatography traps smaller proteins and elutes larger molecules of protein in the resin
• Column Chromatography uses glass/plastic tube with resin and buffer solution
• Ion exchange chromatography attracts the opposite charge of the protein
• If a positively charged resin traps/attracts negatively charged proteins, positively charged proteins are eluted/separated (ion Exchange Chromatography)
• In a high salt conc., it can compete with the resin for the bound proteins (negatively charged proteins can now be eluted) - Ion Exchange Chromatography
• Affinity Chromatography uses the Lock-and-Key Binding wherein protein binds to a molecule with specific affinity (ligand) to carry out their biological activity
• Affinity Chromatography is used to separate larger quantities of proteins and antibodies
• Molecular Separation of Protein Molecules includes electrophoresis and column chromatography
• SDS-PAGE - Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
• SDS disrupts the structure of protein to be linear and binds most protein and breaks disulfide bonds
• SDS-PAGE uses polyacrylamide gel
• Sample in SDS-PAGE run from negative to positive charge
• Advantages of Using SDS

  1. Coats all polypeptides with negative charges
  2. Mask the natural charges of subunits
• The number of SDS molecules bound to a polypeptide is proportional to the length of the polypeptide
• Colorimetric staining methods detect 10-1 ng of protein bonds
• Coomassie blue staining is an organic dye for protein detection in SDS-PAGE gel

  • Advantage: Quantitative binding of the dye to the protein, low price, and good reproducibility
  • Disadvantage: Long staining time, low sensitivity, and narrow dynamic range
• Zinc-reverse staining uses imidazole and zinc salts for protein detection and compatible with MASS spectrometry

  • Based on the precipitation of zinc imidazole in the gels
• Silver staining is based on the binding of silver ions to the protein followed by the reduction to free silver sensitization and enhancement
• Silver staining 


  bands are dark brown to black in a light beige background
• Use glutaraldehyde-free modified silver staining instead of common silver staining wince proteins are differentially sensitive to silver ions
• Fluorescence Staining Methods:

  • CyeDyes
  • NileRed
  • SyPro
  • Epicocconone
• 2-D Gel electrophoresis separates complex protein sample based on the isoelectric point and size
• Isoelectric focusing is used wherein proteins are electrophoresed in a narrow tube gel containing ampholytes that sets up a pH gradient
• Isoelectric focusing

  As proteins migrate towards the increasing pH, they will stop migrating once they reached their isoelectric point
• During 2-D Gel Electrophoresis, the strip is placed on top of the SDS-PAGE and sealed with agarose
• After 2-D electrophoresis, identify the proteins through MASS spectrophotometry
• Western Blotting-laboratory method used to detect specific protein molecules from among a mixture of proteins
• Solid support used in Western Blotting

  • Nitrocellulose
  • Charge or uncharged nylon sheet
  • Polyvinylidene difluoride (PVF)
• Western blotting mark the target proteins using primary and secondary antibodies for visualization
• Diffusion-method of protein transfer done by capillary action (put weights/use vacuum on the membrane for a faster capillary action)
• Diffusion-A steady flow of the buffer through the gel and membrane for a rapid transfer
• Electroblotting - proteins are driven/transferred by an electrical field

  • Immerse the gel and membrane in a tank field with buffer 
• Blocking - step in western blotting to lessen non-specific binding of antibodies & improves the sensitivity of the assay by adding BSA or milk
• BSA-bovine serum albumin (blocks non-specific binding site in western blot)
• • Reacted antibodies that are bound to proteins can be visualized and identified
• • Antibodies are conjugate to HRP (horseradish peroxidase) and alkaline phosphatase that produces color emission